Discriminating Interpatient Variabilities of RAS Gene Variants for Precision Detection of Thyroid Cancer

Key Points Question Is discrimination of interpatient variabilities of RAS gene variants associated with improved accuracy in malignancy diagnosis among thyroid nodules? Findings This diagnostic study of 620 patients, including 438 surgically resected thyroid tumor tissues and 249 thyroid nodule fine-needle aspiration biopsies, delineated interpatient differences in RAS variants at the variant allele fraction (VAF) levels, ranging from 0.15% to 51.53%. While RAS variants alone, regardless of the extent of variation, were associated with low-risk thyroid cancer in 88.8% of tumor samples, they did not definitively distinguish malignancy of an unknown tumor; however, detection of interpatient variabilities of RAS, BRAF, and TERT promoter variants in combination could assist in classifying indeterminate thyroid nodules. Meaning These findings suggest that discrimination of interpatient differences in genomic variants could facilitate precision cancer detection, including preoperative malignancy diagnosis and stratification of low-risk tumors from high-risk ones, among patients with indeterminate thyroid nodules.


Introduction
Thyroid cancer, especially papillary thyroid cancer (PTC), has experienced a rapid increase in incidence since the 1980s 1 and is primarily diagnosed through ultrasonographic examinations and fine-needle aspiration (FNA) biopsy of suspicious nodules. 2,3However, approximately 30% of FNAs exhibit an indeterminate diagnosis, and 10% of findings are nondiagnostic. 4Patients with indeterminate thyroid nodule findings usually undergo diagnostic surgery, with 20% to 30% of nodules being detected as malignant.Thus, up to 70% to 80% of patients with indeterminate nodules found histologically benign have undergone unnecessary surgical procedures.Patients with nondiagnostic cytological findings are typically recommended for a repeat FNA, with 13% of nodules detected as being malignant. 4Cancer arises along with genetic alterations.Molecular assays of FNA specimens are being increasingly used to enhance preoperative diagnostic accuracy for patients with indeterminate cytological findings and avoid unnecessary surgery for benign thyroid nodules. 2,5S is the most frequently variated gene family in human cancer.Approximately 19% of patients with cancer harbor activating variations from 3 RAS gene isoforms: NRAS (OMIM 164790) in 17% of patients, HRAS (OMIM 190020) in 7% of patients, or KRAS (OMIM 190070) in 75% of patients. 6][9][10] In thyroid tumors, RAS gene variations are detected in tumors spanning a wide spectrum of histological diagnoses, with a prevalence of 10% to 30% in PTC, [11][12][13] 40% to 50% in follicular thyroid carcinomas (FTCs), 14,15 12% to 85% in follicular adenoma or hyperplasia, and 5% to 46% in noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTPs). 14,16Indeterminate thyroid nodules carrying RAS variants have shown malignancy rates varying from 9% to 83%, [7][8][9][10]17 and such discrepancies can be primarily attributed to the use of small patient cohorts in these studies. Desite the widespread application of RAS variants in panel tests, assays of RAS variants often yield inconclusive results in detecting malignancy of thyroid nodules, frequently leading to a diagnostic surgery.2,12,18 On the contrary, BRAF V600E and TERT promoter variants (C228T and C250T) are the most frequently detected genetic variants in thyroid nodules, providing a more definitive basis for cancer diagnosis.[19][20][21] Interpatient variabilities in genomic variants may reflect differences in tumor statuses among individuals.20 However, the diagnostic impact of discriminating interpatient variabilities of RAS variants on cancer detection remains unclear, particularly under the 2022 updated fifth World Health Organization (WHO) classification of thyroid neoplasms.22 In alignment with the WHO classification, the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC) 23 has updated nomenclature for each of the 6 diagnostic categories: I, nondiagnostic (ND); II, benign; III, atypia undetermined significance (AUS); IV, follicular neoplasm (FN); V, suspicious for malignancy (SFM); and VI, malignant.23 Currently, the methods of detecting RAS variations are mainly based on polymerase chain reaction (PCR) and Sanger sequencing or next-generation sequencing (NGS).This

Methods
This prospective diagnostic study was reviewed and approved by the Sinai Health Research Ethics Bord.All patients provided written informed consent, and samples were deidentified for data analysis.Data are reported in alignment with the Standards for Reporting of Diagnostic Accuracy (STARD) reporting guideline.

Patients and Clinical Samples
A total of 438 thyroid tissue specimens were obtained from surgically resected thyroid tumors with a maximum dimension of

Droplet dPCR Assays of RAS, BRAF V600E, and TERT promoter variants
Molecular assays for the most prevalent RAS variants of 3 RAS genes, NRAS (Q61R or Q61K), HRAS (Q61R or Q61K), and KRAS (G12C, G12D, G12V, G12A, or G13D), were developed using locked nucleic acid probe-based droplet dPCR by following the strategy and procedures recently established for the VAF assays of BRAF V600E and TERT promoter variants (C228T and C250T). 20,26The details of DNA extraction, dPCR assays, and verification of RAS variants using PCR and Sanger sequencing were documented in the eMethods in Supplement 1.

Statistical Analysis
Data were summarized as frequencies and percentages for categorical variables and means and SDs for continuous variables.Blinded central review-based 2022 WHO histologic classification and 2023 BSRTC were used as the reference standard.

Interpatient Variabilities of NRAS, HRAS, and KRAS Variants in Thyroid Tumors
Molecular VAF assays were developed for the quantitative detection of RAS variants at singlenucleotide resolution positive for NRAS, HRAS, and KRAS in tumor tissues but not in the adjacent healthy tissue, which were verified by Sanger sequencing (eFigure 1 in Supplement 1).Of 438 tumors that underwent surgery, 89 (20.3%) were identified with the presence of RAS variants, including 51 (11.6%) with NRAS, 29 (6.6%) with HRAS, and 9 (2.1%) with KRAS variants, in mutually exclusive existence from each other (Figure 1 and Table 1).When compared with the 3 RAS gene isoforms across all tumor subtypes, the profiles of interpatient variabilities were delineated at the VAF levels ranging from 0.15% to 51.53%, specifically from 0.59% to 51.53% for NRAS, from 0.36% to 43.56% for HRAS, and from 0.15% to 46.64% for KRAS variants, with no significant difference among 3 isoforms (P = .16)(Figure 1; eFigure 2 in Supplement 1).Of these variants, 84 (94.4%) exhibited a VAF of greater than 1% and 5 showed a VAF of less than 1%, with 1 NRAS, 2 HRAS, and 2 KRAS variants.

Discussion
In this diagnostic study, interpatient variabilities in RAS variants were delineated in thyroid tumors with VAFs ranging from 0.15% to 51.53% using sensitive VAF assays.While RAS variants alone, regardless of the VAF levels, were associated with thyroid cancer in 88.8% of thyroid nodules harboring such variants, they did not definitively distinguish malignant tumors from NIFTP and  benign ones.However, they did facilitate the stratification of low-risk tumors from high-risk ones among malignant neoplasms.Furthermore, interpatient differences in the 5 gene variants were discriminated in 42.9% of indeterminate FNAs, 23.7% ND FNAs, and all FNAs with follow-up surgical pathology-confirmed malignancy.
Currently, molecular assays of RAS variants do not effectively risk stratify tumors due to their limited sensitivities and specificities. 27,28In our study, the sensitive VAF assays identified substantial interpatient differences in the most common RAS gene variants, including 57.3% of NRAS variants in predominance, 33.7% of HRAS variants, and 9.0% of KRAS variants.In a comparable PTC cohort from The Cancer Genome Atlas study, the prevalence of RAS variants was 12.9% in PTCs, including 8.5% with NRAS, 3.5% with HRAS, and 1.0% with KRAS, based on NGS assays. 11In contrast, our study observed a prevalence of 23.1% for RAS variants in PTCs, classified by the 2017 WHO classification, 29 including 13.5% with NRAS, 7.4% with HRAS, and 2.2% with KRAS (eFigure 3 in Supplement 1), suggesting that VAF assays revealed higher frequencies of RAS variants in thyroid neoplasms.Hence, significant discrepancies from different methods of detecting genomic variants may result in falsenegative results or missed diagnoses of clinical significance, particularly when methods with lower sensitivities are used. 28,30In addition, a high agreement observed in VAF assays between residual FNA biopsies and matched surgical specimens underscores the clinical significance of using residual specimens.At a direct cost of $12.36 per laboratory-developed test reaction coupled with a turnaround time within 8 hours from specimen receipt to result (eTable 4 in Supplement 1), this approach facilitates the timely and rapid delivery of molecular results concurrently with cytological examination on the same source biopsies, holding promise as an effective addition to existing protocols for personalized thyroid cancer care.
High rates of RAS variants were identified in lesions exhibiting follicular architecture, such as NIFTP (41.7%) and IEFVPTC (50.7%).It is noteworthy that 70.6% of CPTCs carrying RAS variants exhibited a predominantly follicular growth pattern, with most of them presenting encapsulation.
Unfortunately, discriminating variant differences did not improve the stratification power of RAS variants in distinguishing between malignant neoplasms and NIFTPs, follicular adenomas, or oncocytic adenomas, nor between lesions exhibiting differential follicular architecture, such as NIFTP and IEFVPTC neoplasms.The limited effectiveness of RAS variants in stratifying these histological types may be attributed to their close similarity in gene expression profiles. 27,31,32Moreover, low VAF events of RAS variations, including those at VAF less than 1%, were associated with an equally high risk of cancer as high VAF events.This finding aligns with that of a 2017 study that reported an equivalent malignancy rate in RAS variants detected at VAF less than 10% compared with variants detected at VAF greater than 10%. 8[35] The widespread implementation of molecular assays as routine cancer diagnosis remains a challenge.7][38] RAS variants alone, including the low VAF events, do not confirm the malignancy of an unknown tumor; therefore, they should not solely dictate clinical decisions. 39However, RAF variants do enhance the stratification of low-risk tumors, 12,27 aiding in informing the extent of operation.Second, BRAF V600E and TERT promoter variants were detected exclusively in malignant tumors and exhibited a stronger association with aggressive tumor behaviors, aligning with our prior findings and those of other studies. 20,21,40,41The inclusion of RAS variants into BRAF V600E and TERT promoter variant assays significantly enhanced the sensitivity for malignancy detection, albeit with a trade-off of reduced specificity.In addition, RAS variants coexisting with BRAF V600E and/or TERT promoter variants tend to be enriched in high-risk cancers, such as thcPTC, FTC, OCA, ATC, and MTC.
1 cm or larger from 436 consecutive patients who underwent surgery between February 1, 2016, and April 4, 2022, and 249 FNA specimens were collected from 234 consecutive patients who underwent biopsy procedures between January 22, 2020, and March 2, 4,23, at Mount Sinai Hospital, Sinai Health, Toronto, Canada.All surgical tissue specimens sampled were quickly placed in liquid nitrogen and transferred to −80 °C for long-term preservation.As for preoperative biopsies, all FNAs were routinely obtained under ultrasonographic guidance using a 23-gauge needle and subjected to CytoLyte (Hologic) fixation.After cytological examination according to the BSRTC,4,23the leftover materials of a total of 249 FNA biopsies were collected and stored at 4 °C until DNA purification.These preoperative biopsies primarily included ND and indeterminate (BSRTC categories I, III, IV, and V) specimens, along with some malignant (BSRTC category VI) and benign (BSRTC category II) specimens.A follow-up of thyroid nodules was conducted among patients who had previously undergone FNA procedures and subsequently underwent surgery.The patient clinical